The long-term goal of this application is to determine the mechanism of action of topoisomerase II (TOP2)-directed anticancer drugs. In addition to their antitumor activities, TOP2-directed anticancer drugs;uch as VP-16 have been associated with the development of therapy-related acute myelogenous leukaemia (AML). Indeed, our preliminary studies have shown that VP-16 behaves as a stage I tumor promoter in the mouse skin carcinogenesis model. TOP2-directed anticancer drugs are known to trap both TOP2 isozymes, TOP2 alpha and TOP2 beta, into their respective covalent complexes with DNA known as cleavable or cleavage complexes. These two TOP2 isozymes are regulated differently during the cell cycle and most likely perform different functions. However, the differential roles of these two TOP2 isozymes in the antitumor and carcinogenic activities of TOP-directed anticancer drugs remain unclear. Our recent studies have demonstrated that TOP2 beta but not TOP2 alpha cleavage complexes induced by the TOP2-directed anti-cancer drug, VP-16, trigger ubiquitin/26S proteasome-dependent degradation of TOP2 beta (TOP2beta down-regulation). This is the first demonstration of a TOP2 isozyme-specific cellular response to TOP2-directed anticancer drugs. TOP2beta down-regulation is dependent on RNA transcription but not new protein synthesis. Concurrent with TOP2 beta down-regulation, the large subunit of RNA polymerase II, but not other proteins, is also degraded by 26S proteasome. These and other results have suggested that a ubiquitin/26S proteasome pathway(s) is activated locally in the vicinity of the arrested RNA polymerase elongation complexes, leading to the degradation of the TOP2 beta-DNA covalent complex. We further hypothesize that degradation of the TOP2 beta-DNA covalent complex results in exposure of theTOP2-concealed double-strand breaks, which could contribute to both tumor cell death and DNA sequence rearrangements/carcinogenesis. Elucidation of the differential cellular responses to TOP2 cleavage complexes formed by TOP2 isozymes may form the foundation for future development of TOP2 isozyme-specific anticancer drugs. The objective of the current application is to characterize the ubiquitin/26S proteasome pathway induced by TOP2-directed anticancer drugs and to explore the biological consequences of TOP2 beta down-regulation in tumor cell death and carcinogenesis.